Abstract: Bacteriocins are a large class of ribosomally synthesized toxins that have been shown to be effective antibiotics, food preservatives, anti-infectives and anti-cancer therapeutics. It is believed that about half of all bacteria and archaea produce at least one bacteriocin, and current efforts in genome analysis will likely lead to a tremendous increase in the number and diversity of this class of antibiotics. Many of these bacteriocins have been isolated as natural products;thus little is known about how they are biosynthesized. We have developed the first in vitro method to reconstitute the highly potent bacteriocin Streptolysin S (SLS). Reconstitution of the SLS toxin has demonstrated that a precursor peptide and three conserved enzymes work in concert to produce an active toxin. We have furthermore demonstrated for the first time that the synthetase enzyme complex is capable of accepting non-cognate substrates. We propose that this system can therefore be used with a library of substrates to generate entirely novel artificial peptide bacteriocins, many of which will undoubtedly have therapeutic value. Many of these compounds are likely to be potent antibiotics;importantly, many of these will have other pharmacological and therapeutic uses that we have not yet uncovered. The work detailed herein proposes a wide-scale approach for the biosynthesis and screening of this class of novel bacteriocins. First, we will produce a library of novel bacteriocins using our reconstitution method whereby the synthetase complex converts a library of precursors to active compounds. Second, we will develop an integrated high-throughput screening approach to discover activities of these novel bacteriocins. These aims will not only lead to a tremendous increase in the discovery of novel antimicrobial and therapeutic compounds, but will for the first time initiate an integrated approach to the utility of engineered bacteriocins in general. Public Health Relevance: Antibiotic resistance is one of the most important health concerns today, as the current arsenal of antibiotics is rapidly becoming ineffective. This proposal presents a method of generating an entirely new class of antibiotic compounds, many of which will undoubtedly have tremendous public health value.